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THE ANATOMICAL RECORD 216~1-9(1986)
Ultracytochemistry of Ouabain-Sensitive
K+-Dependent p-Nitrophenyl Phosphatase in
Rat Incisor Enamel Organ
PHILIAS R. GARANT AND TAKAHISA SASAKI
Department of Oral Biology and Pathology, School of Dental Medicine, Health Sciences
Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8700
ABSTRACT
Sprague-Dawley strain rats of 4-5 weeks old were perfusion-fixed
with either a mixture containing 0.1 or 0.25%glutaraldehyde and 2%formaldehyde,
or a 2% formaldehyde in 0.1 M sodium cacodylate buffer for 10 minutes. Nondecalcified 30-50-pm sections of the enamel organ taken from lower incisors were
then processed for ultracytochemical demonstration of ouabain-sensitive, K -dependent, p-nitrophenyl phosphatase, by use of the one-step lead method, representing
the second dephosphorylative step of Na+-K+-ATPase.Throughout the secretory,
transition, and maturation stages of amelogenesis, the enzymatic activity was demonstrated along the cytoplasmic side of the plasma membranes of the stratum
intermedium and the papillary layer cells, especially along their numerous microvilli. The plasma membranes forming gap junctions and desmosomes were free of
reaction or showed slight focal precipitates of reaction products. The stellate reticulum and the outer enamel epithelium exhibited either a weak reaction or were
reaction negative. Secretory ameloblasts showed a weak trace-like reaction along
the basal and lateral cell surfaces; however, the latter surfaces were sometimes
completely free of reaction. Tomes’ processes were usually reaction negative. Ameloblasts in the transition and maturation stages were devoid of enzymatic activity,
except for a slight reaction along the plasma membranes of the basal cell surfaces of
transition ameloblasts facing the papillary layer. The enzymatic activity described
above was completely dependent on the presence of potassium and substrate in the
incubation media and was almost completely inhibited by an addition of 10 mM
ouabain to the incubation media,
+
Enamel maturation involves the removal of protein
and water from young enamel concomitant with crystal
growth to form the hardest substance in the body (Deakins, 1942). It has been demonstrated that a large portion
of enamel matrix proteins undergo rapid breakdown and
removal after secretion (Seyer and Glimcher, 1977;
Glimcher et al., 1977; Robinson et al., 1981; Robinson et
al., 1982; Nanci et al., 1985). The removal of matrix
breakdown products and water is thought to be under
the control of secretory and maturation ameloblasts and
the overlying papillary layer cells (Reith, 1970; Reith
and Cotty, 1967; Kallenbach, 1966, 1968; Garant and
Nalbandian, 1968; Garant, 1972; Josephsen and Fejerskov, 1977; Sasaki, 1984a,b;Nanci et al., 1985).
Most investigators of enamel organ structure agree
that during enamel maturation the papillary layer and
maturation ameloblasts take on the appearance of a
transport epithelium (Kallenbach, 1966, 1968; Garant
and Nalbandian, 1968; Garant, 1972; Reith, 1970). This
suggests that the movement of water and solutes out of
the enamel may not be due solely to the permeability
and porosity of the enamel organ and the enamel, but
also to an active cellular process.
No clearly defined hypothesis of how the enamel organ
might function as a transporting epithelium capable of
0 1986 ALAN R.LISS, INC.
effecting water and solute movement out of the enamel
has as of yet been put forth. In this report, we demonstrate the ultrastructural localization of Naf-K+ATPase on the plasma membranes of the stratum intermedium and the papillary layer cells, and we propose
that local osmotic gradients created in the enamel organ
might provide a driving force to move water and solutes
out of the maturing enamel.
MATERIALS AND METHODS
Sprague-Dawley strain rats, 4-5 weeks old, were anesthetized by an intraperitoneal injection of sodium pentobarbital and fixed by transcardiac perfusion with one
of the following fixatives for 10 minutes at room temperature: (1) a mixture containing 2% formaldehyde and
0.25% glutaraldehyde in a 0.1 M sodium cacodylate
buffer (pH 7.4); (2) a mixture containing 2% formaldehyde and 0.1%glutaraldehyde in the same buffer; and
(3) 2% formaldehyde in the same buffer. After fixation,
the fixative was immediately washed out by perfusion
with 0.1 M sodium cacodylate buffer for 5 minutes, then
the lower incisors were dissected out of the jaws and
Received January 8,1986; accepted April 15, 1986.
2
P.R. GARANT AND T. SASAKI
ULTRACYTOCHEMISTRY OF Na+-K+-ATPaseIN RAT INCISOR ENAMEL ORGAN
placed in a 0.01 M sodium cacodylate buffer containing
0.25 M sucrose and 10% dimethylsulfoxide (DMSO;
Sigma Chemical Company), pH 7.4,overnight. DMSO is
a selective activator of the K+-stimulated phosphatase
and shifts its pH optimum to 9.0 (Albers and Koval,
1972).Nonfrozen sections of the enamel organ of 30-50pm thickness were prepared using a vibratome (Oxford
Vibratome Sectioning System) without any decalcification of the tissues.
For ultracytochemical demonstration of the enzyme,
the sections were incubated for 10 min at room temperature (about ZZOC)in a medium (Mayahara et al., 1980)
consisting of 250 mM glycine-KOH buffer, pH 9.0,10
mM p-nitrophenyl phosphate (magnesium salt; Sigma
Chemical Company), 25% DMSO, 4.0 mM lead citrate
(dissolved in 50 mM KOH), and 2.5 mM levamisole.
Final pH was adjusted to 8.8 to 9.0 using 10 M KOH. To
ensure substrate specificity, ouabain sensitivity, and K+
dependency, the sections were incubated in one of the
following media: (1) standard medium described above
lacking substrate (p-nitrophenyl phosphate); (2) standard medium containing 10 mM ouabain; and (3) standard medium, in which K + was replaced by Naf
(glycine-KOH buffer was replaced by glycine-NaOH
buffer). I n these experiments, we used freshly prepared
solutions for incubation, and all incubation media were
clear of any precipitates.
After incubation, the sections were rinsed with 0.1 M
sodium cacodylate buffer (pH 7.4) several times and postfixed with 1.5% potassium ferrocyanide-reduced 1% osmium tetroxide for 30 minutes a t 4°C. They were then
block-stained with 1% uranyl acetate in 10% ethanol for
30 minutes at room temperature, dehydrated through a
graded ethanol series and propylene oxide, and embedded in Poly Bed 812.To avoid a dislocation of reaction
products, tissue processing from fixation to pre-embedding in a 1:l mixture of propylene oxide and Poly Bed
812 were carried out within 2 days. Thin sections were
cut using a diamond knife on a n LKB ultratome 111and,
without any additional staining, were examined with a
JEOL lOOB electron microscope at 60 kV.
Fig. 1 . The proximal portion of secretory ameloblasts (Ab) and the
stratum intermedium cells (SI). Reaction products of lead phosphates
are seen along the plasma membranes of these cells. Inset figure shows
a higher magnification of microvilli of the stratum intermedium cells.
Precipitates of reaction products are visible along the cytoplasmic side
of the plasma membranes but not in the extracellular spaces. The
plasma membranes forming gap junctions (GJt and desniosome (D) are
reaction negative or show a slight focal reaction. Mt, mitochondria.
x 17,000;inset, ~ 4 5 , 6 0 0 .
Fig. 2. The outer enamel epithelium (OEE) and adjacent fibroblast
F b ) showing no enzymatic reaction. EM, extracellular matrix of surrounding connective tissue. ~ 2 2 , 8 0 0 .
Fig. 3. The supranuclear region of secretory ameloblasts. Very slight
reaction products are seen along the plasma membranes (indicated by
arrows). The Golgi membranes (Go) and secretion granules (SG) are
free of reaction. ~ 3 4 , 2 0 0 .
Fig. 4. The Tomes process (TP) of secretory ameloblast showing no
enzymatic reaction. x 34,200.
Fig. 5. The proximal parts of the Tomes processes (TP). The enamel
matrix of the interrod growth region contains reaction precipitates
(indicated by arrows), but the cytoplasmic side of the plasma membranes is reaction negative. ~28,500.
3
RESULTS
Secretory Stage
In the enamel organ at the stage of enamel secretion,
a n enzymatic reaction was observed mainly along the
cytoplasmic side of the plasma membranes of the stratum intermedium cells, especially along their microvilli.
Reaction precipitates of lead phosphates were usually
adjacent to the plasma membranes or within the subsurface cytoplasm, but were not observed in deeper cytoplasm nor in the extracellular spaces. Gap junctions
were usually free of reaction, while desmosomal membranes occasionally showed slight reaction precipitates
(Fig. 1). Enzymatic activity along the plasma membranes became weaker towards the stellate reticulum
and the outer enamel epithelium, which seldom showed
a positive reaction (Fig. 2).Secretory ameloblasts exhibited a weak reaction in the plasma membranes of the
basal cell surfaces facing the stratum intermedium cells,
and less frequently along their lateral cell surfaces. Cytoplasmic organelles and secretion granules were always reaction negative (Fig. 3). In Tomes’ processes,
both the plasma membranes and cytoplasmic inclusions
showed no enzymatic reaction. Adjacent newly secreted
matrix of rod- and interrod-growth regions sometimes
contained scattered reaction precipitates; but the plasma
membranes themselves were free of reaction (Figs. 4,5).
TransitionStage Between Enamel Secretion and Maturation
At the transition stage, the enamel organ consisted of
transition ameloblasts and the papillary layer cells. Enzymatic reaction was observed mainly along the cytoplasmic side of the plasma membranes of the papillary
layer cells. The plasma membranes forming gap junctions and desmosomes between papillary layer cells and
between papillary layer cells and ameloblasts were usually reaction negative. Cytoplasmic matrix and cell organelles such as mitochondria, Golgi membranes, and
endoplasmic reticulum were free of reaction (Fig. 6). The
papillary layer cells at this stage sometimes contained
large phagosomes containing the remnants of various
cell organelles; these were also reaction negative (Fig.
7).The transition ameloblasts showed no enzymatic reaction along their plasma membranes, except at the
basal cell surfaces facing the papillary layer cells (Figs.
63). Although cell organelles and secretion granules of
transition ameloblasts were always free of enzymatic
reaction, some membrane-bounded bodies resembling
phagosomes contained a considerable amount of reaction product (Fig. 9).
Maturation Stage
At the stage of enamel maturation, the enamel organ
consisted of ruffle-ended and smooth-ended ameloblasts
and the papillary layer cells. Many fenestrated capillaries were situated between the epithelial ridges of the
papillary layer. A most intense enzymatic activity was
found along the cytoplasmic side of the plasma membranes of papillary layer cells, especially along their
numerous microvilli (Fig. 10). Gap junctions and desmosomes were reaction negative. Cytoplasmic matrix and
cell organelles of papillary layer cells showed little or no
enzymatic reaction except in regions subadjacent to the
plasma membrane (Fig. 10). Both ruffle-ended and
smooth-ended ameloblasts showed no enzymatic reac-
4
P.R. GARANT AND T. SASAKI
ULTRACYTOCHEMISTRY OF Na+-K+-ATPaseIN RAT INCISOR ENAMEL ORGAN
tion in their cell organelles and along their plasma
membranes (Figs. 11,12>.The endothelial plasma membranes of capillaries in the papillary layer were usually
reaction negative (Fig. 13, inset).
We found little enhancement or reduction of enzymatic
activity and no change in enzyme localization among
the three different fixatives employed in this study (Figs.
13,141.
Control Experiments
When p-nitrophenyl phosphate was omitted from the
incubation medium, no precipitated reaction products
were observed in any cell organelles or plasma membranes of papillary layer cells and other enamel organ
cells (Figs. 15,16). Substitution of K+ with Na+ and
addition of 10 mM ouabain into the incubation media
produced a strong inhibition of the enzymatic reaction
(Figs. 17,18). Only slight focal precipitates of lead
phosphates were still observed in the subsurface cytoplasm of papillary cells when K+ was substituted for
Na+ (Fig. 17).
DISCUSSION
Biochemically, Mornstad (1978) demonstrated the
presence of a ouabain-sensitive, K+-stimulated, p-nitrophenyl phosphatase in the homogenates of the rat incisor enamel organ. This hydrolytic reaction represents
the second step of ATP dephosphorylation by Na+-K+ATPase (Skou, 1974; Ernst, 1975). In Mornstad's experiment, only 30% of the enzyme activity was inhibited by
ouabain. Furthermore, because of the use of tissue homogenates, it was impossible to associate the enzyme
activity with any defined cytological structures. In 1980,
Mayahara et al. developed a one-step lead method for
the electron microscopic localization of ouabain-sensitive, K f -dependent, p-nitrophenyl phosphatase: i.e., the
equivalent of Naf-Kf-ATPase. This is the method we
have used to localize Na+-K+-ATPaseat the ultrastructural level in the enamel organ.
The present observations demonstrate the site of Na K+-ATPase, and presumably that of a sodium pump,
throughout the stratum intermedium and the papillary
layer cells, and although somewhat weaker, along the
basal surfaces of secretory and transition ameloblasts.
Localization of reaction products at the cytoplasmic side
of the plasma membranes indicates the internal release
of the hydrolyzed phosphates by Na+-K+-ATPaseand,
by extrapolation, the transport of sodium across the
+
Fig. 6.The proximal portion of transition ameloblast (Ab) and adjacent papillary cells (PC). Reaction precipitates are visible along the
plasma membranes of these cells excepting gap junctions (GJ) and
desmosomes 0).~28,500.
Fig. 7. Large electron-dense phagosome in a papillary cell, which
contains no reaction products. X 17,000.
Fig. 8.The distal portion of transition ameloblasts in the regions
close to the maturation ameloblast layer and to the secretory ameloblast layer (inset). Transition ameloblasts in both regions show no
enzymatic activity. x 17,000; inset, ~22,800.
Fig. 9. The supranuclear cytoplasm of transition ameloblasts. Membrane-bounded lysosomal bodies contain reaction products in their
matrix. Inset figure shows a higher magnification view of a lysosomal
body containing reaction products and many vesicular and tubular
structures. Go, the Golgi membranes. ~28,500;inset, ~57,000.
5
membrane to the extracellular milieu (Ernst, 1975;
Mayahara et al., 1980; Ueno, et al., 1984).
Considerable evidence has been accumulated in various kinds of epithelial cells that a Na+ pump, namely
Na+-K+-ATPase (E.C. 3.6.1.3.), functions in reabsorption, exchange, and active transport of solutes across the
plasma membranes CBonting, 1970; Blaustein, 1974;
Leuenberger and Novikoff, 1974; Ernst, 1975; Hoshi et
al., 1976; Berman et al., 1977; Mills and DiBona, 1977,
1978; Ueno et al., 1984). Water movement is thought to
be a secondary consequence of active sodium transport
across the plasma membranes (Curren and MacIntoch,
1962; Diamond and Bossert, 1967; Mills and DiBona,
1980). Most epithelia that engage in transport of water
and solutes through the active maintenance of Nat
pump-generated osmotic gradients possess the barrier of
zonulae occludentes across the distal (i.e., luminal) ends
of the cells (Claude and Goodenough, 1973). The intercellular barrier at this location may be total or partial
(Machen et al., 1972; Martinez-Palomo and Erlij, 1975).
In epithelia that are involved in isosmotic reabsorption
of water and solutes such as the colon and the gall
bladder, the barrier is not fully sealed (Machen et al.,
1972). In these leaky epithelia, tracers such as lanthanum penetrate through the zonula occludens junctions, and the intercellular space is a pathway of lowered
electrical resistance (Machen et al., 1972). The tall columnar cells that make up these epithelia possess Na+K'-ATPase along their basolateral surfaces (i.e., opposite the end with the zonula occludens junction), and
thus are thought to be able to pump Na+ into the basolateral intercellular spaces creating a local osmotic gradient (DiBona and Mills, 1979). Despite the fact that the
luminal zonulae occludentes are leaky, gradients are
believed to be maintained because of the complexity of
the basolateral intercellular space and the amplification
of the plasma membrane that borders this space (Welling et al., 1978; Welling and Welling, 1976). In fact, the
leakiness of the zonula occludens may account for the
high volume of water that moves across these epithelia
(Machen et al., 1972). As Na+ is pumped across the
basolateral plasma membrane, the intracellular level of
sodium is replenished by passive entry of Na+ across
the luminal surface of the cell.
The structure of the enamel organ possesses some
severe problems to a conceptualization of how it might
function as a transport epithelium. First of all, the
enamel organ is made up of several cell layers. Ameloblasts border onto the enamel and the stratum intermedium, and papillary layers are located between the
ameloblasts and the blood supply to the enamel organ
(Kallenbach, 1967;Garant and Nalbandian, 1968;Reith,
1970; Garant, 1972). To our knowledge, only frog skin
epithelium possessed Na+-K+-ATPase at the inwardfacing membranes, different from other nonstratified
epithelia (Mills and DiBona, 1977).
A second difficulty is presented by the fact that not all
ameloblasts have distal zonulae occludentes. Secretory
ameloblasts have two sets of tight junctions at both the
proximal and distal ends of cell bodies, and only the
latter one is considered to be a zonula occludens (Warshawsky, 1978; Sasaki et al., 1982). The proximal tight
junction has been shown to be permeable to horseradish
peroxidase (Kallenbach, 1980b; Sasaki, 1984a).Maturation ameloblasts exist in cohorts of ruffle-ended @A) and
smooth-ended (SA) cell types along the surface of the
6
P.R. GARANT AND T.SASAKI
ULTRACYTOCHEMISTRY OF Na+-K+-ATPaseIN RAT INCISOR ENAMEL ORGAN
enamel (Josephsen and Fejerskov, 1977; Reith and
Boyde, 1979). A well developed zonula occludens junction, made up of about ten rows of tight junctional
strands, is present on the distal end of RA and on the
proximal end of SA (Sasaki and Garant, in press). On
the other hand, a permeable intercellular space is present at the proximal end of the RA and the distal end of
the SA (Josephsen and Fejerskov, 1977; Kallenbach,
1980s; Sasaki et al., 1983; Sasaki and Garant, in press).
Because of the different location of the zonulae occludentes in RA and SA, an open pathway is believed to
exist from the enamel space to the papillary layer. This
pathway is an indirect one from the enamel beneath the
SA, proceeding along the lateral intercellular spaces to
the region of RA, where access to the papillary layer
intercellular spaces is gained through the open proximal
borders of the RA (Kallenbach, 1980a). A bidirectional
exchange of substances is thought to occur along this
pathway. Intravenously injected tracers such as horseradish peroxidase have been shown to gain access to the
enamel at the location of the SAs (Kallenbach, 1980a;
Takano and Ozawa, 1980; Sasaki et al., 1983).
The concentration of Na+-K+-ATPaseon the plasma
membranes of the cells of stratum intermedium and
papillary layer suggests that if an osmotic gradient is
established in the enamel organ, it is within the intercellular spaces of the stratum intermedium and papillary layers. Thus, it would seem that the positioning of
a zonulae occludentes in the secretory and maturation
ameloblasts at either the distal or the proximal ends of
these cells would work equally well in establishing a
barrier between the stratum intermedium and papillary
layers and the enamel. If these zonulae occludentes are
leaky, water would be drawn across the junctions from
the enamel to the stratum intermedium and the papillary layers. Kallenbach (1980a) and Sasaki (1984) documented that, at least in early secretion zone, the distal
zonula occludens of secretory ameloblast is permeable to
molecules as large as those of horseradish peroxidase
(molecular weight 43,000). Movement of water from the
7
enamel might drag matrix breakdown products out of
the enamel toward the secretory and maturation ameloblasts. Cytochemical analysis of rat incisor enamel
indicated that a relatively steep rise of mineral content
was observed when it was expressed in terms of tissue
volume, suggesting the replacement of matrix by water
(Hiller et al., 1975). They also described that the subsequent increase in Ca and P per volume of enamel reflected the replacement of water as further mineral was
acquired. In an earlier chemical study of developing pig
molar enamel, Deakins (1942) demonstrated that Ca
influx into the enamel was compensated chiefly by a
loss of water, which had been preceded by a loss of
organic matrix. Accordingly, water movement across the
enamel organ probably mediated by Na+-K+-ATPase
may be a necessary process for the replacement of matrix and, eventually, water during progressive enamel
mineralization. We visualize a net flow of water across
the enamel toward the papillary layer and vasculature.
Such a flow would not preclude diffusion of substances
from ameloblasts into the enamel, nor from the pulpal
blood vessels into the enamel.
In other transport epithelia, such as small intestine,
renal proximal tubule, and gall bladder, sodium enters
the cells passively at the Iuminal surfaces (Blaustein,
1974;Hoshi et al., 1976; DiBona and Mills, 1979).IfNa+
were to enter the secretory and maturation ameloblasts
passively at the distal (i.e., next to the enamel) surfaces,
it could move down a concentration gradient across the
extensive gap junctions between the ameloblasts and
the cells of stratum intermedium and papillary layers.
The colloid osmotic pressure of serum within the extensive, closely juxtaposed, and fenestrated capillaries could
serve as a sink to remove water and small molecular
weight substances from the intercellular spaces of the
papillary layer (Garant and Gillespie, 1969; Sasaki et
al., 1984; Sasaki and Garant, in press).
ACKNOWLEDGMENTS
We wish to thank Mrs. Kris Vandenberg for her typing
of the manuscript. This study was supported by grant
DE 05586 to Dr. P.R. Garant from the National Institute
of Dental Research.
Fig. 10. The papillary layer cells in the transition stage showing
reaction precipitates along the cytoplasmic side of the plasma memLITERATURE CITED
branes. The plasma membranes forming gap junctions (GJ) and desmosomes (D) contain few reaction products. Inset higher magnification Albers, R.W., and G.J. Koval (1972) Na+-K+-activated adenosine triphosphatase. VII. Concurrent inhibition of Na+-K+-adenosinetrifigure shows a localization of reaction precipitates in the microvilli of
phosphatase and activation of K+-nitrophenylphosphatase
papillary cells. x 17,000; inset, ~ 4 5 , 6 0 0 .
activities. J. Biol. Chem., 2473088-3092.
Fig. 11. The proximal (inset) and distal portions of ruffle-ended mat- Berman, A.L., A.M. Azimora, and F.G. Gribakin (1977) Localization of
Na+, K+-ATPase and Ca++-activatedMg+ +-dependentATPase in
uration ameloblasts (Ab) having the ruffled borders (RB) and adjacent
retinal rods. Vision Res., 17527-536.
papillary cells (PC). Enzymatic reaction is seen along the plasma
membranes of papillary cells’ microvilli and of the ameloblast’s basal Blaustein, M.P. (1974) The interrelationship between sodium and calcium fluxes across cell membranes. Rev. Physiol. Biochem. Pharcell structures. GJ, gap junctions. x 17,000.
macol., 70:33-82.
Fig. 12. The proximal (inset) and distal portions of smooth-ended Bonting, S.L. (1970) Sodium-potassiumactivated adenosinetriphosphatase and cation transport. In: Membrane and Ion Transport, Val. 1.
maturation ameloblasts (Ab), which lack both the ruffled border and
E.E. Bittar, ed. New York, Wiley-Interscience, pp. 257-368.
the distal junctional complexes, and adjacent papillary cells (PC). Enzymatic reaction is seen prominently in the papillary cells and weakly Claude, P., and D.A. Goodenough (1973) Fracture faces of zonulae
occludentes from “tight” and “leaky” epithelia. J. Cell Biol.,
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transporting epithelia. Fed. Roc.,38134-143.
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8
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Fig. 15. The papillary cells incubated in the medium lacking substrate, p-nitrophenyl phosphate. No reaction precipitates are seen in
either the plasma membranes or the cytoplasm. ~ 2 8 , 5 0 0 .
Fig. 16. The ruffled border zone (RBI of maturation ameloblasts
incubated in the medium lacking substrate, which is clear of any
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Fig. 17. The papillary cells incubated in the medium, in which K +
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~28,500.
Fig. 18. The papillary cells incubated in the medium containing 10
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- the -plasma mem-
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9
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